Tandem mass spectrometry, or MS/MS, is a well known technique used to improve a spectrometer's signal-to-noise ratio and which can provide the ability to unambiguously identify analyte ions. Whilst the signal intensity may be reduced in MS/MS (when compared with single stage MS techniques), the reduction in noise level is much greater.
Tandem mass spectrometers have been used to analyse a wide range of materials, including organic substances such as pharmaceutical compounds, environment compounds and biomolecules. They are particularly useful, for example, for DNA and protein sequencing. In such applications there is an ever increasing desire for improving the analysis time. At present, liquid chromatography separation methods can be used to obtain mass spectra of samples. LC techniques often require the use of “peak-parking” to obtain full spectral information and there is a general consensus among persons skilled in the art that the acquisition time needed to obtain complete information about all peaks in a mass spectrum adds a significant time burden to research programs. Thus, there is a desire to move to higher throughput MS/MS.
Structure elucidation of ionised molecules can be carried out using tandem mass spectrometry, where a precursor ion is selected at a first stage of analysis or in a first mass analyser (MS1). This precursor ion is subjected to fragmentation, typically in a collision cell, and fragment ions are analysed in a second stage analyser (MS2). This widely used fragmentation method is known as collision induced dissociation (CID). However, other suitable dissociation methods include surface induced dissociation (SID), photo-induced dissociation (PID) or metastable decay.
Presently, there are several types of tandem mass spectrometer geometries known in the art in various geometric arrangements, including sequential in space, sequential in time, and sequential in time and space.
Known sequential in space geometries include magnetic sector hybrids, of which some known systems are disclosed in Tandem Mass Spectrometry edited by W F McLafferty and published by Wiley Inter-Science, New York, 1983; quadrupole time-of-flight (TOF) spectrometer described by Maurice et al in Rapid Communications in Mass Spectrometry, 10 (1996) 889-896; or TOF-TOF described in U.S. Pat. No. 5,464,985. As described in Hoagland-Hyzer's paper, Analytical Chemistry 72 (2000) 2734-2740, the first TOF analyser could be replaced by a separation device based on a different principle of ion mobility. The relatively slow time-scale of precursor ion separation in an ion mobility spectrometer allows the acquisition of a number of TOF spectra over each scan. If fragmentation means are provided between the ion mobility spectrometer and the TOF detector, then all-mass MS/MS becomes possible, albeit with very low precursor ion resolution.
Sequential in time mass spectrometers include ion traps, such as the Paul trap described by March et al in Quadrupole Storage Mass Spectrometry published by John Wiley, Chichester, 1989; or FTICR spectrometers as described by A G Marshall et al, Optical and Mass Spectrometry, Elsevier, Amsterdam 1990; or LT Spectrometers such as the one disclosed in U.S. Pat. No. 5,420,425.
Known sequential in time and space spectrometers include 3D trap-TOF (such as the one disclosed in WO 99/39368 where the TOF is used only for high mass accuracy and acquisition of all the fragments at once); FT-ICR such as the spectrometer disclosed by Belov et al in Analytical Chemistry, volume 73, number 2, Jan. 15, 2001, page 253 (which is limited by the slow acquisition time of the MS2); or LT-TOF spectrometers, (for example as disclosed in U.S. Pat. No. 6,011,259, which transmits only one precursor ion but which the inventors claim to have achieved a 100% duty cycle).
All of these existing mass spectrometers are only able to provide sequential analysis of MS/MS spectra, that is, one precursor mass at a time. Put another way, it is not possible to provide an all-mass spectra for all precursor masses in a single analysis using these existing mass spectrometers. Insufficient dynamic range and acquisition speed of MS-2 mass spectrometers are considered to be a limiting factor in the spectrometer's ability.
This dynamic range and acquisition speed problem has been partially addressed for Fourier Transform ion cyclotron resonance (FTICR) mass spectrometers, as described in Analytical Chemistry, 1990, 62, 698-703 (Williams E R et al) and in the Journal of the American Chemical Society, 115 (1993) 7854, Ross C W et al. Two different multiplex approaches have been demonstrated which take advantage of a multi-channel arrangement. These are as follows:
Two Dimensional Hadamard/FTICR Mass Spectrometry
In this method, a sequence of linearly independent combinations of precursor ions are selected for fragmentation to yield a combination of fragment mass spectra. Encoding/decoding of the acquired “masked” spectra is provided by Hadamard transform algorithms. Williams E R et al (referred to above) have shown that for N different precursor ions, a given signal to noise ratio could be achieved in experiments having a reduced spectra acquisition time of N/4-fold.
Two Dimensional Fourier/FTICR Mass Spectrometry
This method uses an excitation waveform to excite all the precursor ions. This provides different excitation states for different masses of precursor ions. Using stored waveform inverse Fourier Transform (SWIFT) methods, the excitation waveform is a sinusoidal function of precursor ion frequency, with the frequency of the sinusoidal function increasing from one acquisition to another. As a result, the intensities of fragment ions for a particular precursor ion are also modulated according to the applied excitation. Inverse 2D Fourier Transform applied to a set of transients results in a 2D map which unequivocally relates fragment ions to their precursors.
According to Marshall A G (referred to above) the first method requires substantially less data storage and the second method requires no prior knowledge of the precursor ion spectrum. However, in practical terms, both methods are not compatible with commonly used separation techniques, for instance HPLC or CE. This is due to the relatively low speed of FTICR acquisition (which is presently no faster than a few spectra per second), and a relatively large number of spectra required. Also, unless the LC separation method is artificially “paused” using relatively cumbersome “peak parking” methods, the analyte can exhibit significant intensity changes within a few seconds (in the most widely used separation methods). Further, the use of peak parking methods can greatly increase the time to acquire spectra.
GB-A-2,378,312 and WO-A-02/078046 describes a mass spectrometer method and apparatus using an electrostatic trap. A brief description is provided of some MS/MS modes available for this arrangement. However, it does not address any problems associated with all-mass MS/MS analysis in the trap. The precursor ions are ejected from a storage quadrupole, and focussed into a coherent packet by TOF focussing so that the ions having the same m/z enter the electrostatic trap at substantially the same moment in time.
The trajectories of ions in an electrostatic trap are described by Makarov in “Electrostatic Axially Harmonic Orbital Trapping: A High Performance Technique of Mass Analysis”, Journal of Analytical Chemistry, v. 72, p 1156-1162 (2000). From the equations of motion presented in Makarov's paper, it follows that the axial frequency is independent of the energy and the position of ions in the trap (or phase of ions as they enter to trap). Thus, the axial frequency of ion motion is used for mass analysis.